Glycolysis and TCA Cycle - Abali 2/26/16 Flashcards
glycolysis overview
- breakdown of glucose to get energy (ATP)
- conducted by all tissues
all cells pick glucose up via glucose transporters
step 1 from there: phosphorylation of glucose via HEXOKINASE or GLUCOKINASE
types of glucose transporters
active vs facilitated transport
insulin sensitive vs. insulin insensitive
active transport [Na-glucose cotransporters]
- insulin-insensitive
- intestinal epithelium
- renal tubules
- choroid plexus
facilitated transport [dependent on glucose conc gradient]
- insulin-sensitive [GLUT4]
- sk muscle, adipose tissue (“need insulin 4 GLUT4 in the 4 muscle/fat limbs”)
- insulin-insensitive
- most tissues [liver, brain, RBCs, etc]
GLUT4 mobilization
insulin dependent
muscles/adipose tissue
in absence of insulin, GLUT4 is sequestered intracellularly in vesicles
1. insulin binds to cell membrane receptor, upregulates recruitment of GLUT4 to cell membrane
- GLUT4 increases insulin-mediated uptake of glucose into cell
* glucose phosphorylated to keep it trapped in cell - when insulin drops, GLUT4 moves back into intracellular storage pool for recycling
* vesicles fuse to form endosome
key GLUT transporters
- relative Km
- site of action
GLUT1 : low Km (1)
- basal glucose uptake in RBCs and brain
GLUT2 : high Km (15-20)
- pancreatic beta cells (regulation of insulin)
- liver (storage of excess glucose)
GLUT3 : low Km (1)
- basal glucose uptake in brain neurons
GLUT4 : medium Km (5) - insulin dependent
- sk muscle, adipose tissue
how does a cell hang on to glucose?
“tagging” with ATP makes glucose → glucose-6-phosphate
- G6P v hydrophilic, won’t diffuse out of cell
- catalyzed by glucokinase (liver) or hexokinase (all tissues)
hexokinase vs glucokinase
- location
- relative Km
- inhibition
hexokinase (works at max levels even when glucose low)
- all tissues
- low Km
- G6P feedback inhibition
glucokinase (stores continuously, but best when glucose is high - insulin dep)
- liver
- high Km
- no feedback inhibition
implication: lowish Km of GLUT4 and low Km of hexokinase means muscle/fat are priority, but there is feedback inhibition to prevent them from trapping so much glucose that plasma stores drop
similarly, high Km of GLUT2 and high Km of glucokinase means liver only picks up glucose for storage when there’s enough to go around but lack of feedback inhibition means it can do this continuously (taking in more glucose over time)
de vivo disease
hereditary deficiency of GLUT1
- drop in insulin-indep GLUT1 which picks up glucose in brain
- decreased glucose in CSF
symptoms
- seizures and devpt delay
- neuro symptoms: ataxia, dystonia, dysarthia
tx
- ketogenic diet (high protein/fat) - ketones provide alt energy source for brain in absence of glucose
glycolysis: two phases, three enzymes, net gains
two phases
- “preparation”
6-carbon glucose → 3-carbon glyceraldehyde-3-P
- “payoff”
G3P → pyruvate
three regulated enzymes (kinases)
- gluco/hexokinase
- phosphofructokinase-1 (PFK1)
- pyruvate kinase
net gains
- 10 rxns that turn 6C glucose into 2 x 3C pyruvate
- prep: 2ATP consumed
- payoff: 4ATP + 2NADH produced
- most rxns are reversible (except for the ones regulated by the three enzymes)
3 key regulated rxns of glycolysis
- regulation by hormones
in general
- insulin: upregulates glycolysis (fed state)
- glycogen: downregulates glycolysis (fasted state)
glucokinase: glucose → G6P
PFK1: fructose-6P → fructose-1,6-bisphosphate
pyruvate kinase: phosphoenolpyruvate → pyruvate
regulation of hexokinase vs glucokinase
hexokinase
- feedback inhibition by G6P
glucokinase
- inhibited by F6P : gets GK transported into nucleus and sequestered there via binding to GKRP (GK reg protein)
- inhibition reversed either by high intracell glucose or high intracell F1P
- expression of GK increased by insulin
PFK1
regulates first committed step of glycolysis
fructose6P → fructose1,6bisP
- allosteric enzyme : regulated by many factors
regulation differs in muscle and liver…
- high glucose: lots of PFK1 activity in liver
- energy demads: lots of PFK1 activity in muscle
allosteric regulation of PFK1
“high energy” molecules inhibit PFK1
- ATP
- citrate
“low energy” molecultes activate PFK1
- AMP
-
fructose-2,6-bisphosphate also activates PFK1 [middleman for hormonal regulation]
- F6P → F2,6BP via PFK2
- PFK2 is upregulated by insulin via dephos, downregulated by glucagon via phos
- F2,6BP activates PFK1 to keep glycolysis moving (F6P → F1,6BP via PFK1)
- F6P → F2,6BP via PFK2
how do we achieve the differential regulation of PFK1 in muscle and liver tissue?
distinct PFK2 enzymes present in muscle and in liver
- liver PFK2 follows regular hormonal reg (insulin upreg via dephos, glucagon downreg via phos)
-
muscle PFK2 dependent on allosteric reg by accumulated AMP during exercise
- ensures that skeletal stores of ATP, glycogen will be replenished regardless of glucose status broadcasted by hormones
generation of NADH
occurs during conversion of glyceraldehyde3phosphate → 1,3bisphosphoclycerate [via glyceraldehyde3Pdehydrogenase]
energy generation through substrate level phosphorylation
substrate level phos = direct transfer of P from a substrate to ADP/GDP (as opposed to ATP gen via oxphos]
1,3BPG → 3PG + ATP [via phosphoglycerate kinase]
*can also happen through intermediate conversion to 2,3BPG [R-shifter of Hb dissociation curve!]
- increased glycolysis increases 2,3BPG leading to increased O2 delivery to tissues!
energy generation via pyruvate formation
phosphoenolpyruvate → pyruvate + ATP [via pyruvate kinase]
- second instance of substrate-level phosphorylation in glycolysis
regulation of pyruvate kinase
allosteric regulation
- feed forward activation by fructose 1,6-bisphosphate
hormonal regulation
- insulin upreg
- glucagon downreg
fates of pyruvate
cells w mitochondria and O2?
TCA cycle, ATP gen
cells without mitochondria or lacking O2?
lactate gen, regen of NAD+
- allows another round of glycolysis and gen of 2ATP
- can lead to transient lactic acidosis
anaerobic glycolysis
lactate formation
- major fate of pyruvate in tissues lacking mito or w lousy vasc (lens/cornea of eye, kidney medulla, RBCs)
in exercising muscle: conversion of pyruvate → lactate
- allows glycolysis to continue by recycling NADH → NAD
in liver, heart: NADH is low
- so lactate → pyruvate, NAD → NADH
MODY
maturity-onset diabetes of young
monogenic - traceable to individ mutation
- auto dominant disorder : mutations of glucokinase cause 10-65% MODY
- mild diabetes, only rarely complicated, often treated with meal planning only
pyruvate kinase deficiency
- second most common cause of hemolytic anemia
RBCs lack mitochondria → are completely dep on glucose and glycolysis for egy needs
- use glucose to maintain Na/K ATPase → keeps osmotic balace which keeps cell from swelling/lysing → hemolytic anemia
- before lysis, see distorted cell membranes - characteristic spiculated appearance
- decrease in RBCs = decrease in O2 delivery = buildup of glycolytic intermeds like 2,3BPG
causes of 2,3BPG buildup
consequence
2,3BPG causes O2 diss curve to shift RIGHT
- better delivery of O2 to tissues
causes
- decreased RBC count (hemolytic anemia)
- smoking (compensates somewhat for decreased O2 due to CO)
- altitude acclimatization
- COPD
fluoride and glycolysis
fluoride and phosphate complex together, competitively inhibit enolase → stop glycolysis
[used to stabilize glucose in blood specimens]
TCA cycle overview
occurs in mitochondria, generates energy through oxidation of acetylCoA [derived from carbs, fats, proteins] → CO2 + H2O + ATP
essential/conserved (metabolic defects are v rare)
functions
- generate free energy for ATP synthesis
- fats, carbs, proteins are oxidized to CO2 → produce NADH and FADH2 for oxphos → ATP and H2O
- interconversion of fuel ⇔ metabolites
- first intermediate: acetyl CoA - pdt of many catabolic pathways
energy yield of acetyl CoA
fats, carbs, amino acids are metabolized to acetyl CoA
each acetyl CoA yields:
- 3 NADH (x3ATP)
- 1 FADH2 (x2ATP)
- 1 GTP (=1ATP)
1 acetyl CoA = 12 ATP
anapleuritic reactions
rxns involving catabolism + anabolism
ex. TCA cycle
- TCA cycle generates various intermediates for cellular metabolic pathways
- i.e. it’s important to “refill” these intermediates so the cycle can run to completion
- anapleuritic rxns “fill in” the TCA cycle
implication
- TCA intermediate levels rise and fall depending on needs of cell
- needs of cell will influence TCA cycle via shifts in intermediate pool levels
TCA intermediates and fates
oxaloacetate: intermediate of gluconeogenesis
citrate, oxaloacetate, acetylCoA: membrane transport mechs
alpha ketoglutarate, oxaloacetate: transamination rxns
pyruvate keys: aerobic fed conditions
aerobic fed conditions
- pyruvate → acetyl CoA [pyruvate dehydrogenase complex]
- pyruvate → alanine [pyridoxal phosphate-dependent alanine aminotransferase - remember B6!]
pyruvate keys: aerobic fasting conditions
aerobic fasting conditions
- pyruvate → oxaloacetate [biotin-dependent pyruvate carboxylase]
pyruvate keys: anaerobic conditions
anaerobic conditions
- pyruvate → lactate [lactate dehydrogenase]
- in O2-deprived muscle: pyruvate → alanine [alanine aminotransferase]
pyruvate dehydrogenase complex
bridge between carbs and TCA cycle
PDC converts pyruvate → acetyl CoA, which is used in TCA cycle
complex of 3 enzymes: E1, E2, E3
- each is critical to complex fx
- each has specific cofactors - deficiency of cofactor can produce pathology
E1
pyruvate dehydrogenase component
24 chains
prosthetic: TPP
catalyzes: oxidative decarboxylation of pyruvate
E2
dihydrolipoyl transacetylase
24 chains
prosthetic: lipoamide
catalyzes: transfer of acetyl group to CoA
E3
dihydrolipoyl dehydrogenase
[also in alphaketoglutarate dehydrogenase complex and branched chain alpha-ketoacid dehydrogenase complex]
12 chains
prosthetic: FAD
catalyzes: regen of oxidized form of lipoamide
req coenzymes for PDH complex activity
deficiency issues
B1 (thiamine) : thiamine pyrophosphate (TPP)
B5 (pantothenic acid) : CoA
lipoic acid
B2 (riboflavin) : flavin adenine dinucleotide (FAD)
B3 (niacin) : nicotinamide adenine dinucletide (NAD+)
4 vitamin derivatives + lipoic acid (made in sufficient amt in body)
- deficiencies in any of the coenzymes affect ability to get egy from glucose → sk muscle weakness, neurological disease
PDH complex enzyme cofactor requirements
E1 - thiamine pyrophosphate (B1 derivative)
E2 - CoA (B5 deriv), lipoic acid
- resp for step producing CoA [i.e. need for CoA]
E3 - FAD (B2), NAD+ (B3)
B1/thiamine deficiency
B1/thiamine is used by PDH, alphaketoglutarate DH, branched chain alphaketoacid DH, transketolase
- deficiency affects nucleic acid synthesis, energy metabolism
syndromes
- beri beri
- US: most common in alcoholics (poor nutrition, affects of excess alc on ability to absorb/store thiamine)
- dry (muscle wasting, neuropathy) and wet (CHF + edema)
- Wernicke’s encephalopathy
- triad of confusion, opthalmoplegia, ataxia
- Korsakoff’s psychosis
- memory loss, confabulation, personality change
affects of arsenite and mercury
- inhibit enzymes that use lipoic acid as a cofactor (including PDH complex)
- CNS pathologies (ex. “mad as a hatter”!)
- tx: BAL (British anti-Lewisite) - heavy metal chelator
arsenic poisoning
tasteless, odorless white powder
affects E2 subunit of PDH complex (and other enzymes using lipoic acid as coenzyme)
signs
- PDC deficiency (lactic acidosis, neuro disturbances)
- garlic breath, “rice-water” stools that are bloody, vomiting
fun fact: Van Gogh’s emerald green paint had As might have contributed to his mental episodes
allosteric regulation of PDH complex
high energy moleculte inhibit PDH
- ATP
- acetyl CoA
- NADH
covalent modification : regulation of PDH complex
PDH kinase phosphorylates PDH → inactivates PDH
- activated by high energy molecules
PDH phosphatase dephosphorylates PDH → activates PDH
- activated by insulin in adipose tissue
- activated by Ca in muscle tissue
dichloroacetic acid
synthetic analog of pyruvate
- can bind to PDH kinase, inhibiting its action
- prevents inhibition of PDH by keeping it in active dephos form
*might be effective treatment for lactic acidosis or MELAS, but clinical trials havent shown it
regulation of PDH complex
allosteric
- high energy mols inhibit
covalent
- dephosphorylation (PDH phosphatase) activates
- phosphorylation (PDH kinase) inhibits
- high energy mols inhibits
PDH complex deficiency
- metabolic and neurologic defects
- delayed devpt and reduced muscle tone
- often associated with ataxia and seizures
- some have congenital malformation of brain
- most commonly due to mutation in X-linked E1 gene
will see accumulation of pyruvate and lactate, but normal pyruvate : lactate ratio
- glycolysis occurs, but can’t do anything with the pyruvate except reduce it into lactate
tx
- ketogenic diet, severe restriction of protein and carb : improved mental devpt
- ensures that cells use acetyl CoA from fat metabolism
- if mutation affects binding of thiamine to E1, might try high dose thiamine supplementation
metabolic and neurologic conseqs of PDH compex deficiency
metabolic : lactic acidosis (pyruvate → lactate)
neurological : hypotonia, poor feeding, lethargy, seizures, mental retardation
3 enzymes sharing E3…
- PDH complex
- alpha ketoglutarate DH
- branched chain alpha ketoacid DH
Leigh disease
group of disorders characterized by lactic acidosis (rare)
- defects in PDH and alpha ketoglutarate complexes → cant catabolize BCAAs
- incrased levels of lactate, alpha ketoglutarate, BCAAs
- tissues dependent on aerobic metabolism (brain, muscle) most severely affected
- impaired motor fx, neuro dorders, mental retardation
TCA cycle overview
- occurs in mitochondria
- all organs except ones without mito go through it
- runs during fasted and fed states
- aerobic
- step 1: condensation of acetyl CoA + OAA → citrate
- OAA regenerated at end of cycle
- produces 3NADH + 1FADH2 + GTP + CO2
irreversible reaction of TCA cycle and enzymes/regulation
- acetyle CoA → citrate
* citrate synthase - isocitrate → alpha ketoglutarate
* isoditrate dehydrogenase - alpha ketoglutarate → succinyl CoA
* alpha ketoglutarate dehydrogenase
regulation via allosteric activation/inhibition [NOT HORMONES]
- HIGH ENERGY mols DEACTIVATE : ATP, NADH
- LOW ENERGY mols ACTIVATE: ADP, CA
role of cellular energy in determining TCA cycle rate
high cellular energy → TCA cycle inhibited
low cellular energy → TCA cycle activated
allosteric regulation of TCA cycle
ATP, NADH, succinyl CoA, fatty acyl derivatives all indicate high energy
- inhibit TCA cycle
ADP indicates low energy
- activates TCA cycle
“Citrate Is Krebs’ Starting Substrae For Making OAA”
stops on the TCA cycle
- citrate
- isocitrate
- alphaKetoglutarate
- succinyl CoA
- succinate
- fumarate
- malate
- oxaloacetate
oxidative decarboxylation in TCA cycle
2 successive decarboxylations via 2 dehydrogenase rxns → release of 2 CO2 + 2 NADH
- isocitrate dehydrogenase
- alpha ketoglutarate dehydrogenase
substrate level phos in TCA cycle
succinyl CoA → succinate
[succinyl CoA thiokinase - takes off the CoA, generates a GTP)
- substrate level phos producing a GTP
3 reversible rxns to wind up TCA cycle
succinate → fumarate
[succinate dehydrogenase - FAD reduced to FADH2 (complex II of etc)]
fumarate → malate
[fumarase - adds a H2O]
malate → oxaloacetate
[malate dehydrogenase - completes recycling process, generates NADH]
net carbon yield of TCA cycle
ZERO
- 2 C introduced as acetyl CoA
- 2 C liberated as CO2
implication: need to provide the other intermediates of the cycle in order for rxn to proceed
- constant input of acetyl CoA would keep the cycle running smooth forever
- TCA cycle doesn’t operate in isolation…
- intermediates are pulled off in other direction/rxns, need to be replaced via anapleuritic rxns [involve intermediates]
energy yield from TCA cycle
3 NADH [NADH → NAD = 3ATP]
1 FADH2 [FADH2 → FAD = 2ATP]
1 GTP [= 1ATP]
12 ATP total
net energy production from aerobic resp
1 glucose
glycolysis: 2 ATP, 2 NADH
- each NADH from glycolysis worth either 2 or 3 ATP depending on the transporter used to shuttle it to mito
2 pyruvate
TCA cycle: 2 GTPv, 6 NADH, 2 FADH2
38 ATP total
